Method for determining DNA fragmentation in microorganisms

Abstract

The invention relates to a method for determining DNA integrity in microorganisms and to a kit for evaluating DNA integrity in same. Since cell death results in DNA fragmentation, the inventive method can be used to determine DNA fragmentation levels in microorganisms clearly, simply, quickly and precisely.

The invention claimed is: 
     
       1. A process for evaluating the DNA integrity of microorganisms comprising the steps of:
 a) immobilizing the microorganism on a slide, without fixing, by means of including it in an inert medium; 
 b) treating with a lysis solution, comprising an ionic protein denaturing detergent, to extract cell walls, membranes and proteins; 
 c) stabilizing the DNA nucleoid of the microorganism on the slide by means of dry heat, comprising incubating the slide with the lysed sample in a microwave oven or drying oven for a time sufficient to stabilize and firmly adhere the DNA nucleoid to the slide; and 
 d) staining and evaluating the DNA integrity. 
 
     
     
       2. A process according to  claim 1 , wherein the ionic detergent is a detergent selected from the group of sodium dodecyl sulfate (SDS), alkylbenzene sulfate, lauryl sarcosine (sarkosyl), hydrated salt of glycolic acid, and mixtures thereof. 
     
     
       3. A process according to  claim 2 , wherein the ionic detergent is sodium dodecyl sulfate (SDS). 
     
     
       4. A process according to  claim 1 , wherein the lysis solution comprises between 0.001 and 2M dithiothreitol (DTT); between 0.001 and 2M 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris); between 0.001 and 2M ethylenediaminetetraacetic acid (EDTA), and between 0.1 and 3% sodium dodecyl sulfate (SDS). 
     
     
       5. A process according to  claim 2 , wherein the lysis solution comprises between 0.001 and 2M dithiothreitol (DTT); between 0.001 and 2M 2-amino-2(hydroxymethyl)-1,3-propanediol (Iris); between 0.001 and 2M ethylenediaminetetraacetic acid (EDTA), and between 0.1 and 3% sodium dodecyl sulfate (SDS). 
     
     
       6. A process according to  claim 3 , wherein the lysis solution comprises between 0.001 and 2M dithiothreitol (DTT); between 0.001 and 2M 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris); between 0.001 and 2M ethylenediaminetetraacetic acid (EDTA), and between 0.1 and 3% sodium dodecyl sulfate (SDS). 
     
     
       7. A process according to  claim 4 , wherein the lysis solution is adjusted to a pH between 6.5 and 10.5. 
     
     
       8. A process according to  claim 5 , wherein the lysis solution is adjusted to a pH between 6.5 and 10.5. 
     
     
       9. A process according to  claim 6 , wherein the lysis solution is adjusted to a pH between 6.5 and 10.5. 
     
     
       10. A process according to  claim 4 , wherein the lysis solution preferably comprises 0.1M dithiothreitol (DTT); 0.01M (hydroxymethyl)-1,3-propanediol (Tris); 0.05M ethylenediaminetetraacetic acid (EDTA) and 2% sodium dodecyl sulfate (SDS). 
     
     
       11. A process according to  claim 10 , wherein the lysis solution comprises 0.1M dithiothreitol (DTT); 0.01M (hydroxymethyl)-1,3-propanediol (Tris); 0.05M ethylenediaminetetraacetic acid (EDTA) and 2% sodium dodecyl sulfate (SDS). 
     
     
       12. A process according to  claim 11 , wherein the lysis solution is adjusted to a pH of about 10 with NaOH. 
     
     
       13. A process according to  claim 1 , wherein the staining of step d) is carried out with a fluorochrome solution. 
     
     
       14. A process according to  claim 1 , wherein the sample containing the microorganisms is included in an inert microgel. 
     
     
       15. A process according to  claim 14 , wherein the sample containing the microorganisms is included in an agarose microgel. 
     
     
       16. A process for evaluating the DNA integrity of microorganisms according to  claim 1 , wherein the evaluation is carried out by means of a direct visual analysis. 
     
     
       17. A process for evaluating the DNA integrity of microorganisms according to  claim 1 , wherein the evaluation is carried out in an automated manner by means of applying software for analyzing digitized images, obtained by means of cameras coupled to microscopy platforms. 
     
     
       18. A process for evaluating the DNA integrity of microorganisms according to  claim 1 , wherein the evaluating in step (d) is carried out with a computer program comprising instructions adapted for carrying out the evaluation of the DNA integrity of microorganisms. 
     
     
       19. The process according to  claim 1 , wherein the stabilizing in step (c) comprises incubating the slide with the lysed sample in a microwave oven at about 500-1000 W for about 3-10 minutes, or in a drying oven at about 8° C. for at least about one hour.